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Antimicrobial peptide with complex biological activity and preparation method and application thereof

A technology of antimicrobial peptides and bacteriostatic agents, applied in the field of antimicrobial peptides and their preparation, can solve problems such as economic losses in the aquaculture industry, and achieve the effects of good agglutinating bacteria, inhibiting fungi, and good application prospects

Active Publication Date: 2019-01-04
INNER MONGOLIA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 20 years, the breeding process of blue crabs has been troubled by the infection of white spot syndrome virus, Vibrio and some other bacteria. economic loss

Method used

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  • Antimicrobial peptide with complex biological activity and preparation method and application thereof
  • Antimicrobial peptide with complex biological activity and preparation method and application thereof
  • Antimicrobial peptide with complex biological activity and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning, recombinant expression and purification of antimicrobial peptide gene Sp-crustin 6 of Scylla pseudocaveus

[0026] The amino acid sequence of the antimicrobial peptide Sp-Crustin 6 with compound biological activity of the present invention is shown in SEQ ID NO.1; the nucleotide sequence encoding the antibacterial peptide Sp-Crustin 6 is shown in SEQ ID NO.2. Antimicrobial peptide Sp-Crustin 6 was recombinantly expressed and purified according to the following steps.

[0027] (1) Use the animal total RNA extraction kit to extract the total RNA of the mixed tissue samples of 5 kinds of quality tissues such as muscle, hepatopancreas, gills, intestines, and eye stalks of the Scylla pseudocarpus, and obtain the total RNA samples of the mixed tissue of the Scylla pseudocarpus .

[0028] (2) Using the total RNA sample obtained in step (1) as a template, and using the specific primer 5'-CTGGGAAGAAATGACATG-3' as the post-primer of the reverse transcription s...

Embodiment 2

[0039] Example 2 Bacterial Binding Effect of Recombinant Scylla pseudophylla Antimicrobial Peptide rSp-Crustin 6

[0040] The concentration obtained in Example 1 was 200 μg·mL -1 The recombinant protein rSp-Crustin 6 solution was used for the binding experiment of common pathogenic bacteria. First, 200 μL of overnight cultured bacterial liquid (respectively Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Vibrio parahaemolyticus, algae Vibrio, Vibrio harveii, Escherichia coli, Candida albicans, Pichia pastoris), with 200μL concentration of 200μg·mL -1 The target recombinant protein solution was incubated at 37°C and 180rpm for 1.5h on a shaking table in the dark, then centrifuged at room temperature at 6000rpm to collect the bacteria, washed 3 times with 1×PBS buffer for 5min each time, and then Wash the cells with 7% SDS solution for 5 minutes, then wash the cell pellet with 500 μL 1×PBS buffer once, centrifuge at room temperature at 6000 rpm to collect the cel...

Embodiment 3

[0042] Example 3 The liquid bacteriostasis of the recombined Scylla pseudophylla antimicrobial peptide rSp-Crustin 6

[0043] The concentration obtained in Example 1 was 200 μg·mL -1 The purpose of the recombinant protein rSp-Crustin 6 solution is to carry out the liquid antibacterial experiment of common pathogenic bacteria, first Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, Vibrio parahaemolyticus, Vibrio alginolyticus, Harvey Vibrio, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Pichia pastoris were routinely cultured overnight as pathogenic bacteria. On the next day, 100 μL of recombinant Scylla syringae antimicrobial peptides diluted 2-fold with 1×PBS buffer The rSp-Crustin 6 protein solution was mixed with 100 μL of the target pathogenic bacteria solution, and placed in the wells of a clean disposable 96-well microplate for incubation. At the same time, GST protein was used as the control group, and each spotting 1...

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Abstract

The invention discloses an antimicrobial peptide with complex biological activity and a preparation method and application thereof, and belongs to the technical field of molecular biology. The antimicrobial peptide is derived from scylla paramamosain, the amino acid sequence of the scylla paramamosain is shown in SEQIDNO.1 and the coding sequence of the scylla paramamosain is shown in SEQIDNO.2. The preparation method of the antimicrobial peptide includes the following steps that the total RNA of the scylla paramamosain is extracted to serve as a template, and retranscribing is performed to obtain cDNA; the cDNA is used as the template for PCR amplification to obtain nucleotide fragments containing the coding sequence of antimicrobial peptides, the amplified product and anexpression vectorare subjected to enzyme digestion, connection and transformation, escherichia coli engineering bacteria which can express recombinant antimicrobial peptides are constructed, the escherichia coli engineering bacteria are subjected to induction culture, and recombinant antimicrobial peptides are expressed. The antimicrobial peptides has good antimicrobial and antiviral activities, can be used forpreparing antimicrobial or antiviral agents, and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an antimicrobial peptide derived from Scylla pseudocarpus with complex biological activity, a preparation method and application thereof. Background technique [0002] On the basis of innate immunity, vertebrates have evolved an adaptive immune system that can be continuously enriched. The emergence of this immune method has enabled organisms to have a wider range of immune forms during the evolution process, and at the same time transformed from a passive defensive immune method to an active immune method. However, for invertebrates, the innate immune system is the only form of immune defense against pathogenic microorganisms such as bacteria, viruses, and parasites. This system mainly includes two types of cellular immunity and humoral immunity. Cellular immunity mainly involves three types: formation of nodules, encapsulation, and phagocytosis. Humoral ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P31/04A61P31/10A61P31/12
CPCA61K38/00A61P31/04A61P31/10A61P31/12C07K14/43509C12N15/70
Inventor 杜志强沈秀丽李新苍王凯
Owner INNER MONGOLIA UNIV OF SCI & TECH
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